Comparative Genomic and Functional Characterization of Two Lytic Bacteriophages Against Antimicrobial-Resistant Escherichia coli

Background/Objectives: Antimicrobial resistance (AMR) in Escherichia coli is a growing public health concern, particularly in regions affected by environmental contamination and poor wastewater management. Data on locally isolated E. coli-targeting phages in Lebanon remain limited. This study aimed to isolate, characterize, and evaluate two lytic bacteriophages against AMR E. coli. Methods: Two phages, EPIMAM01 (gb:PQ493298) and EPIMRB01 (gb:PQ657784), were isolated from untreated sewage in Beirut using E. coli ATCC 25922. Characterization included double-layer agar assays, one-step growth analysis, and stability testing across temperature and pH ranges. Bacteriolytic activity was assessed in planktonic cultures and preformed biofilms. Host range and efficiency of plating (EOP) were evaluated using clinical isolates. Whole-genome sequencing and comparative analyses were performed. Results: Both phages produced clear plaques and showed a latent period of ~40 min. EPIMAM01 had a higher estimated burst size (140 PFU/infected cell) than EPIMRB01 (75 PFU/infected cell). Both phages remained stable between 4–50 °C and within a pH range of 5–10 but showed marked loss of activity at temperatures ≥60 °C and pH ≤3 or ≥12. EPIMAM01 effectively inhibited planktonic growth of E. coli ATCC 25922, whereas EPIMRB01 showed stronger biofilm-disrupting activity against preformed E. coli biofilms. Both phages lysed several of the 17 tested clinical E. coli isolates. Comparative analyses of gene presence/absence patterns, bacterial defense systems, and adsorption phenotypes among the tested E. coli strains identified mlaA, ydcQ, and ompD-2 as candidate adsorption-associated genes and suggested CRISPR systems may reduce susceptibility. Genomic analysis classified both phages as T4-like phages lacking lysogeny, virulence, or AMR genes. Conclusions: EPIMAM01 and EPIMRB01 are lytic phages with complementary antimicrobial properties, supporting their potential for further development as AMR control agents.